All cell lines used in the study were purchased from the American Type Culture Collection. Cells were tested for mycoplasma contamination every 1 month, and only mycoplasma-negative cells were used. Wild-type and Snailfl/fl MMTV-PyMT cancer cells were generated and maintained in our laboratory as described previously (20). MMTV-PyMT cancer cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, #10099-147), EGF (10 ng/ml; PeproTech, #315-09), hydrocortisone (500 ng/ml; Sigma-Aldrich, #H0888), insulin (5 mg/ml; #I9278), cholera toxin (20 ng/ml; #C8052), and 1% penicillin-streptomycin (Thermo Fisher Scientific, #15140122). HEK293T, HCT116, RKO, 4T1, DLD1, SW620, SUM159, and MDA-MB-231 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. For isolation of human BrCa primary cells, freshly isolated breast tumors were rinsed extensively three times in cold phosphate-buffered saline (PBS) supplemented with 1% penicillin-streptomycin and chopped into small fragments (~1 mm3). Tissue fragments were digested into single-cell suspension by incubation in DMEM containing 10% FBS, 1% penicillin-streptomycin, collagenase type 1 (1 mg/ml; Sigma-Aldrich, #C0130), and hyaluronidase (125 U/ml; STEMCELL Technologies, #07919) for 12 to 18 hours at 37°C with slow agitation. After incubating for 5 min at room temperature without agitation, the stromal cell–enriched supernatant was discarded, and the epithelial cell–rich pellets were filtered with a 40-μm nylon mesh to remove cell clumps. Tumor epithelial cells were washed three times, resuspended, and cultured in DMEM/F12 medium containing 5% heat-inactivated FBS, EGF (10 ng/ml), hydrocortisone (500 ng/ml), insulin (5 mg/ml), cholera toxin (20 ng/ml), and 1% penicillin-streptomycin.

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